Abstract
Temperature-dependent DEER effects are observed as a function of methyl rotation by either leucine- or nitroxide-specific protonated methyl groups. Both species induce a site specific enhancement in the apparent Tm-relaxation of the paramagnetic nitroxide label. The presence of a single protonated methyl group in close proximity (4–10Å) to only one of the two nitroxide rotamer ensembles in protein A results in a selective and substantial decrease in Tm, manifested by differential decay of the peak intensities in the bimodal P(r) distance distribution as a function of the total dipolar evolution time, temperature or both. Temperature assisted Tm filtering will capture the DEER structural analysis of biomolecular systems heterogenic conformations, including complexes involving multimeric proteins.